#FEMSmicroBlog: Investigating interactions of membrane proteins in Pseudomonas aeruginosa
Bacterial cell membranes are lipid bilayers with integrated proteins and protein complexes. These membrane proteins are bridges between the inside and the outside of the membrane-encapsulated compartments, the cytosol, and the periplasm. To fulfill their crucial biological functions, membrane proteins often form complexes with other proteins. In the study “Characterization of a soluble library of the Pseudomonas aeruginosa PAO1 membrane proteome with emphasis on c-di-GMP turnover enzymes” in microLife, the authors investigated interactions between membrane proteins of Pseudomonas aeruginosa to better understand the physiology of this opportunistic pathogen, as Anna Scherhag explains for the #FEMSmicroBlog. #FascinatingMicrobes
The lifestyle and biofilm formation of Pseudomonas aeruginosa
The Gram-negative bacterium Pseudomonas aeruginosa is ubiquitous in the environment, in water and soil while it can also infect humans. People with a weakened immune system, such as cancer patients or patients in intensive care units, are particularly affected by infections with this opportunistic pathogen.
P. aeruginosa can colonise burn wounds, the lower respiratory tract, catheters, or implants. Patients with cystic fibrosis often suffer from chronic infections caused by the pathogen.
Treatment of these infections represents a challenge as the bacterium can form stable biofilms in and on the human body. Bacterial cells live within the biofilm in a self-produced polymer matrix that protects them from antibiotics and the host’s immune system.
The formation of a biofilm is tightly controlled: To decide whether it is appropriate to form a biofilm, bacterial cells must be able to sense their environment. For example, they need to know which nutrients are available or which properties the surface has.
For this purpose, membrane proteins play a crucial role: Since they are exposed to the outside of the cell, they help the bacterium react accordingly.
Creating a native library of membrane proteins of Pseudomonas aeruginosa
To investigate membrane proteins, the extraction of proteins from their membrane is a critical step, as extraction can affect the stability of proteins and protein complexes. A relatively new method to extract membrane proteins is the use of so-called nanodiscs. These are synthetic, amphiphilic copolymers consisting of diisobutylene and maleic acid chains.
In this process, disc-shaped structures of the copolymers invaginate into the membrane and extract all membrane proteins. To stabilise the protein complex and thus the interactions with additional proteins after extraction, a belt of the synthetic polymer wraps around both the membrane protein(s) and the lipids.
The study “Characterization of a soluble library of the Pseudomonas aeruginosa PAO1 membrane proteome with emphasis on c-di-GMP turnover enzymes” in microLife used the synthetic copolymer approach to extract membrane proteins to create a soluble membrane-protein library. The extracted proteins and their interaction partners were analysed by mass spectrometry in a global proteomic approach.
The results show good extraction capacity and reproducibility of the polymer nanodisc approach. Identifying over twenty membrane protein complexes of various sizes suggests that this method is gentle enough to preserve membrane protein complexes.
Elucidating the pathogen’s c-di-GMP signalling network
In a targeted approach, the nanodisc library was combined with pulldown assays using the membrane protein NbdA as bait. This phosphodiesterase is a member of the c-di-GMP network and is thus involved in signal transduction during biofilm formation.
While this approach enriched and identified several candidate proteins, bacterial two-hybrid analysis confirmed the interaction of NbdA with only four proteins. These potential new interaction partners are the diguanylate cyclase SadC, the transcription factor CzcR, the type IV ATPase PilB, and a hypothetical protein.
These findings led to new insights into the c-di-GMP network regulating biofilm formation in P. aeruginosa. The study also presents the advantage of native membrane protein libraries to investigate bacterial protein interactions.
- Read the article “Characterization of a soluble library of the Pseudomonas aeruginosa PAO1 membrane proteome with emphasis on c-di-GMP turnover enzymes” in microLife by Scherhag et al. (2023).
Anna Scherhag is a 4th year PhD candidate in the Department of Microbiology at the University of Kaiserslautern. She completed her undergraduate degree as well as her Master’s degree in Biology at the University of Kaiserslautern (Germany). For her PhD project, Anna is working on membrane proteins in Pseudomonas aeruginosa.
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The section #FascinatingMicrobes for the #FEMSmicroBlog explains the science behind a paper and highlights the significance and broader context of a recent finding. One of the main goals is to share the fascinating spectrum of microbes across all fields of microbiology.
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